植物内生真菌是重要的生物资源,来自植物不同组织的内生菌还有一大部分未被人们所认识,该研究通过查阅国内外相关文献,发现内生菌产生的化合物大多具有生物学活性.在此基础上以蛇足石杉(Hu-perzia serrata )为材料,从其茎部分离纯化得到1株内生真菌,提取这株真菌的总 DNA,用 PCR 扩增 ITS 片段,将测序结果在 GenBank 数据库中进行比对,最终鉴定为葡萄座腔菌属(Neofusicoccum sp．).具体方法：(1)对内生真菌 Neofusicoccum sp．F483进行固体培养基发酵,并用混合有机溶剂提取得代谢产物；(2)采用硅胶、Sephadex LH-20凝胶色谱柱层析、半制备 HPLC 等方法对代谢产物的化学成分进行分离；(3)利用理化性质及1 H-NMR、13 C-NMR、ESI-MS 等波谱数据鉴定化合物结构.结果表明：共分离得到8个化合物,经过波谱数据分析并结合文献对照鉴定结构为 fusaproliferin (1)、过氧麦角甾醇(2)、麦角甾醇(3)、1-(furan-2-yl)-2-hydroxyethanone (4)、脑苷脂 C (5)、腺嘌呤核苷(6)、versicolactone B (7)、versicolactone A (8).该研究结果可为进一步从植物内生菌中挖掘有价值的天然活性产物奠定基础,为新的药用植物内生菌资源的开发利用提供理论依据.%As we know,the plant endophytic fungi are some important biological resources,and up to now,a large part of the plant endophytes which came from a variety of plant organizations had not yet been recognized by the peo-ple,and on the basis of checking out relative technical literature reported in both at home and abroad,the great ma-jority chemical constituents produced by the endophytes were found to possess certain biological activity.This thesis was the further search for new active natural products from pteridophyte Huperzia serrate endophytes.H .serrate (Thunb.)Trev .was collected from Xichou County,Wenshan Prefecture,Yunnan Province.A endophytic fungus was isolated and purified from the stem of H .serrate .After extracting total DNA of this strain,internal transcribedspacer (ITS)was amplified by polymerase chain reaction (PCR),then the amplified product was sequenced.Frag-ment sequencing results were compared to the GenBank sequence database,and eventually,this strain was identified as Neofusicoccum and was named as Neofusicoccum sp.F483.In this study,we adopted the following three meth-ods:(1)The endophytic fungus Neofusicoccum sp.F483 was fermented with solid medium,and then,fungi with medium were extracted with mixed organic solvent ,and the secondary metabolites were obtained;(2)The chemical composition of the fermented metabolites were isolated by column chromatography methods including normal-phase silica gel,Sephadex LH-20,and reversed-phase semi-preparative HPLC;(3)The isolated chemical components were identified by their physical and chemical properties,and a combination of spectroscopic techniques including electrospray ionization mass spectrum (ESI-MS)and one-dimentional nuclear magnetic resonance (1 H-NMR,1 3 C-NMR)spectroscopic analysis.The results were as follows:eight compounds were separated and identified from Neo-fusicoccum sp.F483,and their structures were elucidated by analyzing their spectral data and comparing with the rel-evant reported literature.They were identified as follows:fusaproliferin (1),5α,8α-epidiory-(22E,24R)ergosta-6, 22-dien-3β-ol (2),ergosterol (3),1-(furan-2-yl)-2-hydroxyethanone (4),cerebroside C (5),adenosine (6),versi-colactone B (7)and versicolactone A (8).It will lay a foundation for further mining the valuable natural products from plant endophytes,and will provide a theoretical basis for the development and utilization of the new medicinal plant endophytes resources.
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